Application of high throughput technologies to drug substance and drug product development

research papers

Acta Cryst. (2006). D62, 1267–1275 doi:10.1107/S0907444906033555 1267

Acta Crystallographica Section D

BiologicalCrystallography

ISSN 0907-4449

Application of high-throughput technologies to astructural proteomics-type analysis of Bacillusanthracis

K. Au,a N. S. Berrow,a

E. Blagova,b I. W. Boucher,b

M. P. Boyle,b J. A. Brannigan,b

L. G. Carter,a T. Dierks,c

G. Folkers,c R. Grenha,b

K. Harlos,a R. Kaptein,c

A. K. Kalliomaa,b

V. M. Levdikov,b C. Meier,a

N. Milioti,b O. Moroz,b

A. Muller,b R. J. Owens,a

N. Rzechorzek,b S. Sainsbury,a

D. I. Stuart,a T. S. Walter,a

D. G. Waterman,b

A. J. Wilkinson,b K. S. Wilson,b

N. Zaccai,a Robert M. Esnoufa*

and Mark J. Fogga*

aDivision of Structural Biology, University of

Oxford, Wellcome Trust Centre for Human

Genetics, Roosevelt Drive, Oxford OX3 7BN,

England, bThe York Structural Biology

Laboratory, Department of Chemistry, University

of York, Heslington, York YO10 5YW, England,

and cBijvoet Center for Biomolecular Research,

NMR Spectroscopy, Utrecht University

Padualaan 8, 3584 CH Utrecht,

The Netherlands

Correspondence e-mail: [email protected],

[email protected]

# 2006 International Union of Crystallography

Printed in Denmark – all rights reserved

A collaborative project between two Structural Proteomics In

Europe (SPINE) partner laboratories, York and Oxford,

aimed at high-throughput (HTP) structure determination of

proteins from Bacillus anthracis, the aetiological agent of

anthrax and a biomedically important target, is described.

Based upon a target-selection strategy combining ‘low-

hanging fruit’ and more challenging targets, this work has

contributed to the body of knowledge of B. anthracis,

established and developed HTP cloning and expression

technologies and tested HTP pipelines. Both centres devel-

oped ligation-independent cloning (LIC) and expression

systems, employing custom LIC-PCR, Gateway and In-Fusion

technologies, used in combination with parallel protein

purification and robotic nanolitre crystallization screening.

Overall, 42 structures have been solved by X-ray crystallo-

graphy, plus two by NMR through collaboration between

York and the SPINE partner in Utrecht. Three biologically

important protein structures, BA4899, BA1655 and BA3998,

involved in tRNA modification, sporulation control and

carbohydrate metabolism, respectively, are highlighted. Target

analysis by biophysical clustering based on pI and hydropathy

has provided useful information for future target-selection

strategies. The technological developments and lessons

learned from this project are discussed. The success rate of

protein expression and structure solution is at least in keeping

with that achieved in structural genomics programs.

Received 10 March 2006

Accepted 21 August 2006

1. Introduction

The pan-European project Structural Proteomics In Europe

(SPINE; http://www.spineurope.org/) is primarily aimed at

fostering collaborations to yield methods developments,

particularly those relevant to high-value eukaryotic, especially

human, proteins of biomedical importance. However, two

SPINE partners, Oxford and York, have pursued a colla-

borative pilot project on a biomedically important prokaryotic

target, Bacillus anthracis, as a vehicle for the development of

robust technologies for protein expression, crystallization and

structure determination. This paper looks at the developments

driven by this collaboration and analyses trends that have

emerged from the results.

The Oxford Protein Production Facility (OPPF) is funded

by the UK Medical Research Council to pilot high-throughput

(HTP) protein production and crystallization methods. From

the outset, its remit has been to tackle challenging biomedi-

cally relevant proteins, but as the technical platform for

protein production took shape the need emerged to stress-test

the pipeline under true HTP conditions. At the same time, the

York laboratory wanted to use its involvement in SPINE to

investigate more automated methodologies and already had a

long-standing interest and well developed expertise in the

cloning, expression and crystallization of proteins from the

non-pathogenic soil-dwelling bacterium B. subtilis (Lewis et

al., 1998, 2002; Cladiere et al., 2004), with the main areas of

interest including sporulation and its control, ABC transport

systems and two-component signal transduction. To meet the

goals of both laboratories, it was decided to pursue a colla-

borative study of proteins from B. anthracis, a close genetic

relative of B. subtilis, since it is of clear biomedical importance

and the complete genome sequences of both B. subtilis [4100

predicted open reading frames (ORFs); Kunst et al., 1997] and

B. anthracis (5311 predicted ORFs; Read et al., 2003) were

available. B. anthracis, a highly virulent Gram-positive endo-

spore-forming bacterium, is the aetiological agent of anthrax

(Mock & Fouet, 2001), a member of the ‘B. cereus group’ of

group 1 bacilli (Radnedge et al., 2003) and can be isolated

from soil environments as dormant spores able to survive for

decades (Manchee et al., 1981; Turnbull, 2002). The longevity

of B. anthracis spores and relative ease of production gives

them potential as a bioweapon (Inglesby et al., 2002). Anthrax

is normally a disease of grazing herbivores, but it can also

infect other mammals including humans. Infections are

acquired through the inhalation (inhalation anthrax) or

ingestion (gastrointestinal anthrax) of B. anthracis spores, by

spore entry through breaks in the skin (cutaneous anthrax)

and even from insect bites (Mock & Fouet, 2001). The genome

sequences of B. anthracis (Read et al., 2003) and B. cereus

(Ivanova et al., 2003) show clear differences from that of

B. subtilis (Anderson et al., 2005), consistent with the different

ecological niches inhabited by each organism. These differ-

ences combined with comparative functional and structural

analyses of proteins common across the B. cereus group and

those unique to pathogenic species could lead to new non-

antibiotic therapies.

2. Materials and methods

2.1. Target-selection strategies

Development of bioinformatics tools for target selection

formed an early part of the HTP developments at Oxford and

the OPAL and OPTIC resources are described in Albeck et al.

(2006). The OPTIC database was populated with the 5311

predicted B. anthracis ORFs (Read et al., 2003), a resource

used by both Oxford and York to inform the target-selection

process. York enhanced their selection with the ‘YSBL

Structural Genomics Resource’ (Rodrigues & Hubbard,

2003), populated with all B. anthracis ORFs and exploiting

many of the same resources as OPAL and OPTIC.

For York, the target-selection strategy combined devel-

oping ongoing research interests with deliberate selection of

uncomplicated targets to assess the potential of HTP

methodologies. An initial set of 48 targets for use in HTP

structural proteomics was selected as low-hanging fruit by the

following criteria: (i) molecular weight <50 kDa, (ii) candidate

for molecular replacement, (iii) not part of a complex, (iv)

contains no signal peptides or transmembrane regions, (v)

predicted to be soluble and ordered according to PONDR

(Romero et al., 2001). Targets that were members of families

previously studied by York were particularly favoured. A

second set of 96 targets was selected on the basis of three

specific criteria: (i) 24 targets based on the identification of

genes present in pathogenic Bacillus species (B. anthracis and

B. cereus) but not in the genetically close non-pathogenic

organism B. subtilis; (ii) 27 targets having significant biological

interest, having no molecular-replacement model and/or being

essential to the viability of the organism [primarily conserved

hypothetical protein homologues of B. subtilis proteins iden-

tified from the Database of Essential Genes (DEG; Zhang et

al., 2004)]; (iii) 45 targets selected on bioinformatics criteria

similar to those above but including disorder evaluation with

RONN (Yang et al., 2005; see also Esnouf et al., 2006).

Furthermore, sets of 10–20 targets matching existing York

research interests were selected for individual study, such as

hypothetical histidine kinases and representatives of the

glycoside hydrolase, carbohydrate esterase and glycosyl-

transferase families that had not as yet been fully character-

ized.

Since the primary goal for the Oxford B. anthracis study was

to test the OPPF pipeline, a set of 48 proteins from well

conserved protein families were selected since these have

been hypothesized to be most amenable to structural study,

presumably because they represent the physically stable core

machinery of the cell (O. Herzberg, personal communication).

The initial procedure involved selecting a set of 23 diverse

pathogenic bacteria (including both Gram-positive and Gram-

negative bacteria for which full genome sequences were

available1) followed by an exhaustive all-against-all BLAST

alignment (Altschul et al., 1990) for every protein sequence

predicted from these genomes. TribeMCL (Enright et al.,

2002) was then used to cluster the significant hits (BLAST E

value <0.1) into families and B. anthracis proteins were

considered further if they belonged to families conserved

across 22 (239 families) or 23 (203 families) of the bacterial

genomes. The list of possible targets was further filtered in a

similar process to that employed by the York group based on

size, prediction of transmembrane regions and signal peptides

and possibility of structure solution by molecular replacement.

A target-selection meeting finalized the choice of targets and

included a few ‘challenging’ targets such as those for which no

molecular-replacement solution was available and four

conserved hypothetical proteins. As the Oxford pipeline

developed, these targets were revisited and some construct

redesign was attempted for previously unsuccessful targets.

Finally, a second set, this time of 78 targets, was selected based

research papers

1268 Au et al. � High-throughput structural proteomics-type analysis Acta Cryst. (2006). D62, 1267–1275

1 Bacillus anthracis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni,Chlamydia muridarum, Chlamydophila pneumoniae, Clostridium tetani,Deinococcus radiodurans, Escherichia coli, Haemophilus influenzae, Helico-bacter hepaticus, Lactobacillus plantarum, Listeria innocua, Mycobacteriumtuberculosis, Mycoplasma pneumoniae, Neisseria meningitides, Pseudomonasaeruginosa, Salmonella typhi, Staphylococcus aureus, Streptococcus pneumo-niae, Streptomyces coelicolor, Vibrio cholerae, Yersinia pestis.

on biological interest while relaxing the family conservation

criterion slightly. For some targets several constructs were

attempted in parallel, yielding a total of 96 constructs.

2.2. Experimental strategy at York

York has devised a semi-automated ligation-independent

cloning (LIC-PCR or LIC; Aslanidis & de Jong, 1990) strategy

with Escherichia coli expression and non-cleavable N-terminal

His6 tags suitable for the high-throughput cloning and over-

expression of bacterial targets (M. Fogg, manuscript in

preparation; Alzari et al., 2006). The cloning and expression

vector, pET-YSBLIC, was engineered for LIC by directed

mutagenesis of pET-28a (Novagen) that added an N-terminal

tag (MGSSHHHHHH-) directly onto any expressed protein

cloned into the vector. A small number of targets were cloned

into unmodified pET-28a by conventional restriction/ligation

methods.

The typical protein-production strategy is described in

Alzari et al. (2006). Target coding sequences were amplified

from B. anthracis genomic DNA (Ames strain) by PCR,

cloned into pET-YSBLIC and transformed into E. coli

NovaBlue cells (Novagen). Target clones identified by colony

PCR were transformed into E. coli BL21 (DE3), B834 (DE3)

and/or Rosetta2 (DE3) for protein-overexpression trials and

scale-up. Following purification, protein concentrations were

adjusted to 15–20 mg ml�1 for crystallization trials.

Crystallization screening used a Mosquito nanolitre liquid-

handling robot (TTP Labtech), standard sparse-matrix

conditions and CrystalQuick 96-well crystallization plates

(Greiner Bio-One). Where possible, crystals from these trials

were tested for X-ray diffraction in-house. However,

successful crystallization trials were usually scaled up and/or

used as the starting point for optimization/additive screening,

either by hand or using the Mosquito robot, to improve crystal

size and quality. After in-house characterization, the best

crystals were shipped pre-frozen to synchrotrons (ESRF,

Grenoble or SRS, Daresbury) for data collection. Most

structures were solved by molecular replacement.

Nuclear magnetic resonance (NMR) experiments have

augmented crystallographic studies (see also AB et al., 2006).

Initially, 15N-labelled B. anthracis proteins were screened by

recording their 15N-HSQC fingerprint NMR spectra as

described previously (Folkers et al., 2004). However, two

targets that failed to yield protein crystals, BA1655 and

BA5174, proved suitable for structure determination by

solution NMR. NMR conditions were optimized for both

proteins to increase concentration and stability while reducing

both the pH and overall salt conditions. Complete sets of

NMR spectra for assignment and structure elucidation were

recorded on Bruker Avance spectrometers operating at 700

and 900 MHz 1H frequency. These high field strengths offered

crucial gains in resolution for both proteins (see AB et al.,

2006), which display the rather low dispersion of HN and H�

protons typical of all-helical proteins (see York example

structure below).

2.3. Experimental strategy at Oxford

The OPPF protein-production pipeline also uses ligation-

independent methods. The strategy has evolved during the

course of this B. anthracis study partly as a result of the lessons

learned from it and these methods are outlined in Alzari et al.

(2006). Initially, experiments focused on Gateway technology

using the pET15G expression vector (Luan et al., 2004), which

adds a 3C-cleavable N-terminal His6 tag but also a translation

of the attB site giving a total tag length of 38 amino acids that

appeared to restrict protein expressibility/solubility. Subse-

quent work used the pDEST14 expression vector (Invitrogen),

which only adds a minimal non-cleavable N-terminal His6 tag

(MAHHHHHH-) and gave much better levels of soluble

expression. Work on the set of 78 targets has been based on

the ligation-independent In-Fusion system (Clontech) using

in-house modifications of the pET-28a and pTRIEX2

(Novagen) expression vectors (namely pOPINB and pOPINF,

respectively) that can add either N-terminal or C-terminal

His6 purification tags (see Alzari et al., 2006; Berrow et al.,

manuscript in preparation). Target coding sequences were

amplified by PCR directly from B. anthracis genomic DNA

(supplied by York) or indirectly using the GenomiPhi proce-

dure (Dean et al., 2001). Cloning and small-scale expression

trials followed the methods described in Alzari et al. (2006),

with expression scale-up in either E. coli B834 (DE3) or

Rosetta (DE3) cells. Purified proteins were concentrated for

crystallization trials, with pre-crystallization tests (Hampton)

being used to suggest appropriate protein concentrations.

Crystallization screening followed the standard Oxford

sitting-drop protocols for screening and optimization (Walter

et al., 2003, 2005; Brown et al., 2003) using sparse-matrix

conditions and CrystalQuick 96-well crystallization plates

(Greiner Bio-One). Crystals taken from these nanolitre

screens and optimizations were used directly for data collec-

tion, with crystals shipped to synchrotrons (ESRF, Grenoble

or SRS, Daresbury) in plates and mounted at the beamline.

Most structures were solved by molecular replacement,

although selenium multiwavelength anomalous dispersion

(MAD) analysis at BM14, Grenoble was used in some cases.

3. Results

3.1. Structures determined by York

Table 1 summarizes the progress made by York. The

parallel experiments on sets of 48 and 96 targets are at

different stages, with work on the 48-target set largely

completed, whereas work on the set of 96 is ongoing. The high-

resolution limits of collected data sets ranged from 1.6 to

3.5 A. A total of 21 structures have been determined so far:

ten from the set of 48 targets, two from the set of 96 targets,

five from other targets, two structures with ligands and two

determined by NMR.

The efficacy of the LIC method is clearly demonstrated as

46/48 target ORFs were cloned into the pET-YSBLIC vector

after just two rounds of cloning, with 33/46 (72%) expressed in

soluble form and in sufficient yield to enter crystallization

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Acta Cryst. (2006). D62, 1267–1275 Au et al. � High-throughput structural proteomics-type analysis 1269

trials following protein purification. Diffraction-quality crys-

tals were obtained for 18 of these proteins, yielding 12 data

sets with ten new B. anthracis protein structures solved, two of

which were also solved with bound ligands. The ongoing work

with the more challenging set of 96 targets has yielded single-

pass statistics with a subsequent reduction in the number (72/

96; 75%) of cloned ORFs, as only a single round of cloning,

expression and purification has been carried out. However,

soluble expression statistics compare favourably with the first

set, as 38/54 (70%) of targets so far tested have produced

soluble protein expression suitable for crystallization trials. To

date, this has resulted in six diffraction data sets and two new

structures. All other targets, progressed in smaller groups by

individual graduate students, are at various stages of

completion. This disparate target group comprises subsets

selected on the basis of specific biological interest with little

weight given to overall tractability; consequently, it comprises

the most challenging set.

The discrepancy between data sets collected and solved

structures (Table 1) is a consequence of problems of crystal or

data quality, including problems such as twinning, limited

resolution and data incompleteness. The majority of

constructs used and hence proteins crystallized had the non-

cleavable His6 purification tag (MGSSHHHHHH-) attached;

consequently, in order to assess whether poor-quality crystals

were a result of the presence of this tag, a vector pET-

YSBLIC3C was developed containing a 3C protease cleavage

site for tag removal. An assessment of whether this method

significantly improves crystal quality is ongoing, although

based on the Oxford experiences described below we would

expect some benefit.

3.2. Examples

The tRNA-modifying enzyme ThiI from B. anthracis

(BA4899) was selected as a target for structural determination

as it contains a THUMP domain, a predicted RNA-binding

module found in various RNA-modifying enzymes throughout

the three domains of life (Aravind & Koonin, 2001). ThiI is

responsible for the formation of 4-thiouridine at position 8 of

tRNA, a common modification in prokaryotes. This base

forms a covalent photo-cross-link with a cytosine at position

13 of tRNA when irradiated with near-UV light, thus

providing a sensor for such exposure and a trigger for the

subsequent cellular response (Ramabhadran & Jagger, 1976).

Following cloning (pET-YSBLIC), expression and crystal-

lization, the structure of ThiI was solved (Waterman et al.,

2006) in complex with AMP to 2.5 A resolution by MAD

analysis of crystals of a selenomethionine-substituted form of

the protein (Fig. 1a) using data collected on beamline BM14 at

the ESRF, Grenoble. The structure reveals the THUMP fold

to be unrelated to that of previously characterized RNA-

binding domains. In ThiI, the THUMP domain is accompanied

by an N-terminal ferredoxin-like domain. Analysis of

conserved exposed residues indicates that the tRNA-binding

surface is likely to be formed from both domains; however, a

model of the interaction remains elusive. The modified uridine

at position 8 is buried within the core of tRNA in its canonical

L-shaped conformation, implying that structural changes to

the tRNA molecule are necessary to expose it to the PP-loop

pyrophosphatase active site of the enzyme.

Two targets, BA1655 and BA5174, were identified as

members of the Spo0E-like phosphatase family by amino-acid

sequence comparison with the B. subtilis proteins Spo0E,

YnzD and YisI. In B. subtilis, Spo0A is the key regulator of the

sporulation phosphorelay. The threshold concentration of

Spo0A~P required for sporulation is achieved by the activity

of sporulation sensor kinases. Spo0E-like phosphatases

counter this by dephosphorylating Spo0A~P, thereby inhi-

biting sporulation (Ohlsen et al., 1994; Perego et al., 1994).

Whilst crystallization trials with BA1655 and BA5174 failed,

both proteins were considered suitable candidates for struc-

ture determination by NMR, being small (7.5 and 7.9 kDa,

respectively) and highly soluble. The structures were solved in

collaboration with the SPINE partner in Utrecht. Initial15N-HSQC spectra demonstrated a good dispersion typical of

�-helical proteins, with BA1655 exhibiting greater stability

than BA5174, the latter requiring additional temperature and

concentration optimization. Complete sets of NMR spectra

led to structure elucidation of BA1655 (Grenha et al., 2006, in

the press) comprising a dimer in a four-helix bundle and

consisting of two pairs of helices connected by a tight turn

packed in a head-to-tail manner (Fig. 1b), whereas BA5174 is

monomeric and comprises a similar pair of antiparallel

�-helices. These structures indicated that crystallization diffi-

culties probably arose from disorder at the C-terminus. These

tails were too short (13 and 16 residues for BA5174 and

BA1655, respectively) to be reliably predicted as disordered

by the algorithms used during target selection.

3.3. Structures determined by Oxford

A summary of Oxford progress on the first 48 targets and

the current (early) state of progress with the second 78 targets

is provided in Table 1. Overall, 23 structures have been

determined (including complexes with small molecules) for 19

research papers


Table 1Progress on different B. anthracis target sets by the York and Oxfordlaboratories.

Work on the first sets of 48 targets for each laboratory is largely finished andgives an indication of potential eventual success rates. Work on the othertarget sets is ongoing.

ProgressionYork setof 48

York setof 96

Yorkothers

Oxfordset of48

Oxfordset of78

Totalprogress

Selected 48 96 89 48 78 359PCR 48 94 88 47 76 353Expressed 43 54 67 43 28 235In crystallization 33 38 40 41 9 161Crystallized 32 23 11 22 6 94Data sets 12 6 9 17 5 49Structures solved 10 2 5 15 4 36Structures with ligands 2 — — 2 2 6NMR structures — — 2 — — 2Total structures 12 2 7 17 6 44

different target proteins with high-resolution limits ranging

from 1.6 to 3.3 A, but the success rates for the different

protocols explored has varied greatly.

For the pET15G expression vector (long, cleavable

N-terminal tag) cloning from genomic DNA was successful

(41/48; 85%), but the fraction of targets yielding soluble

expression in screening was low (13/41; 32%) and even for

these the expression levels were often less than 5 mg l�1.

Expression of only six targets could be scaled up successfully

for crystallization trials and these trials were set up using both

cleaved and uncleaved proteins. Diffraction-quality crystals

were obtained with three of the cleaved proteins and all

yielded structures. In addition, one target was successfully

cocrystallized with substrate and product, and the structure

determined. The uncleaved protein yielded crystals for only

one protein and these were of poor quality. Work on the set of

48 targets was repeated using the pDEST14 expression vector

to give a combined coverage of targets of 47/48 (98%). The

pDEST14 vectors (short, uncleavable N-terminal purification

tag) were screened for soluble expression on a small scale

(1 ml) using both IPTG and autoinduction (Studier, 2005)

protocols with generally similar results for well expressing

proteins, although for poorly expressing proteins autoinduc-

tion often outperforms the IPTG protocol. Soluble expression

levels were generally much higher from the pDEST14 vectors

compared with the equivalent pET15G vectors (typical yields

in excess of 5 mg l�1) and a total of 33/44 (75%) targets

entered into crystallization trials. Crystals were grown for 18/

33 (55%) of these targets; X-ray data sets were collected for 14

and yielded 11 structures, including one overlap (at lower

resolution) with a target from the pET15G round.

One of the failed targets was used as part of an initial test of

the In-Fusion technology (Clontech) and this yielded good

soluble expression (>5 mg l�1) and a structure at 2.4 A reso-

lution, prompting further evaluation and eventual adoption of

this technology. In-Fusion-based work with some of the

remaining recalcitrant targets from the initial 48 targets

yielded crystals of three new constructs, two medium-resolu-

tion (3–3.5 A) data sets and one structure. As a result of these

successes, In-Fusion technology is also being used for the new

set of 78 targets (some of which are being processed with

multiple constructs, giving a total of 96 constructs), of which 28

have to date yielded soluble expression.

3.4. Example

The structure determination of B. anthracis d-ribulose-

5-phosphate epimerase (BA3998) showed several interesting

features (Carter et al., in preparation). The protein expressed

in soluble form with the long 3C protease-cleavable tag and

was the only target for which (poor-quality) crystals could be

obtained with the tagged protein. After cleavage, however,

good crystals were obtained and X-ray data were collected

extending to 2.2 A resolution for a crystal belonging to space

group P41212 on Station 14.1 at the SRS, Daresbury. An

attempted soak with d-ribulose-5-phosphate was ultimately

unsuccessful, but improved native data extending to 2.05 A

were collected on beamline ID14EH1 at the ESRF, Grenoble.

The structure of the substrate-free epimerase was solved by

molecular replacement to reveal a hexamer arranged as a

triangular prism (Fig. 1c; R factor = 0.208; Rfree = 0.236).

Slowly growing crystals were obtained from cocrystallizations

with the same substrate and after an initial 2.9 A resolution

data set confirmed the presence of bound substrate and/or

product, a data set extending to 2.3 A resolution was recorded

on beamline ID14EH2 at the ESRF from a cocrystal that took

more than six months to appear and belonged to space group

P212121. This structure was solved by molecular replacement

starting from the substrate-free structure and revealed

d-ribulose-5-phosphate (probably mixed with the product,

d-xylulose-5-phosphate) bound to differing extents in the six

active sites of the hexamer along with catalytic zinc ions (R

factor = 0.211; Rfree = 0.266). These data are the first for a

substrate/product-bound epimerase and allow us to modify

and update the previously suggested model for epimerase

catalysis (Kopp et al., 1999; Jelakovic et al., 2003).

4. Discussion

4.1. Cloning strategies

Traditional methods of cloning using restriction enzymes

require tailoring of the protocol to each individual target and

hence are not amenable to HTP approaches. This has stimu-

lated interest in ligation-independent methods where only the

primers vary between targets. York has developed a custom

LIC-based approach, whereas Oxford has concentrated on

commercial technologies, initially Gateway and more recently

In-Fusion. All methods have proven amenable to parallel-

ization and success rates are high (Table 1) and relatively

independent of target size. Oxford has adapted the cloning

protocols to work effectively on two robotic liquid-handling

stations, the BioRobot 8000 (Qiagen) and the MWG Theonyx

(Aviso GmbH), whereas York are in the process of adapting

their protocols for robotic liquid handling on a Freedom EVO

(Tecan) as part of their newly established High-Throughput

Expression Laboratory (HiTEL). Primer design is also rela-

tively automated; for example, the OPPF primer-design

interface, OPINE, which is built around the Primer3 package,

provides a direct link between the target-selection software

and the LIMS (Albeck et al., 2006). OPINE, along with the

Web Primer resource (http://seq.yeastgenome.org/cgi-bin/

web-primer), was used remotely by York for early primer-

design requirements, although a new primer-design program

developed in-house is now used for all LIC and LIC3C

primers. One advantage of the York and In-Fusion protocols is

that the primers are shorter than those required to produce

the same short-tagged product with Gateway technologies;

furthermore, only one experimental reaction is required,

resulting in significant cost and time savings.

4.2. Expression and purification strategies

Not surprisingly, for bacterial proteins high levels of soluble

expression are routinely observed by York and Oxford using a

research papers


variety of E. coli strains. Purification strategies in both

laboratories are now based on AKTA XPress and Explorer

3D systems (GE Healthcare) and are found to be very effec-

tive. Where the Oxford laboratory requires cleavage of

N-terminal purification tags, this is routinely performed with a

His-tagged 3C protease, which is now seamlessly integrated

into the HTP context and works with high efficiency. The

effect of different tags in expression is discussed in x4.5.

4.3. Crystallization and crystal quality

Oxford has helped pioneer the use of small-volume (100 +

100 nl drop) crystallization trials (Walter et al., 2003, 2005;

Brown et al., 2003) and now has two Cartesian Technologies

MicroSys MIC400 (Genomic Solutions Ltd) robots in heavy

use. York has deployed the Mosquito (TTP Labtech) robot.

Both laboratories have observed an increase in success rate on

reduction of drop volume. Oxford rapidly moved towards a

strategy of optimization using small-volume drops and has

developed standard procedures which have now been imple-

mented on liquid-handling robots and are very effective,

avoiding the need to translate conditions to a larger drop

format (Walter et al., 2005). Crystals from small drops are used

directly for data collection, although these crystals are

frequently too small to be screened using in-house X-ray

facilities. Thus, Oxford has tended to take crystallization trays

to synchrotrons and explore cryopro-

tection strategies on the beamline, an

effective but labour-intensive approach.

In contrast, York has tended to scale up

crystallization-drop volumes during the

optimization process and as a result the

crystals have usually been large enough

for in-house screening; nevertheless,

four of the 21 York structures were

solved using crystals recovered directly

from 96-well screens. The York strategy

allows scaled-up in-house crystals to be

shipped frozen. The effect of the puri-

fication tag on crystal quality is

discussed in x4.5.

4.4. Structure-determination strategies

Most of the targets studied by both

York and Oxford were amenable to

structure determination by molecular

replacement. In general, structure

solutions proceeded in a routine

manner, with automated molecular-

replacement packages, such as CaspR

(Claude et al., 2004) and the CCP4/

eHTPX-supported development of

MrBUMP (Keegan & Winn, manuscript

in preparation; Bahar et al., 2006) often

proving successful. Indeed, several of

the B. anthracis data sets were used to

help in the development and testing of

MrBUMP. Data for one of the Oxford

B. anthracis targets proved particularly

challenging for automated methods and

have helped to drive further develop-

ments. These data (Bahar et al., 2006)

are somewhat unusual in that they are

from a highly mosaic crystal (>2�) that

nevertheless diffracts to high resolution

(1.5 A).

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Figure 1Selected structures of B. anthracis proteins solved by the York and Oxford laboratories. (a) Amonomer of ThiI (BA4899) shown as a ribbon representation. The N-terminal ferredoxin-likedomain is coloured green and forms a continuous �-sheet surface with the THUMP domain (red).The enzymatic PP-loop pyrophosphatase domain is coloured blue and shows the position (as a stickrepresentation) of AMP bound at the active site. The glycine-rich linker joining the THUMPdomain to the PP-loop domain is coloured yellow. (b) A ribbon representation of the lowest energystructure (PDB code 2bzb) of the Spo0E-like phosphatase target BA1655 showing the individualmonomers in green and blue. (c) A ribbon diagram showing the hexameric structure of unligandedd-ribulose-5-phosphate epimerase (BA3998). Individual chains are coloured red, orange, yellow,green, light blue and dark blue. The hexameric structure of the protein with bound substrate hasalso been determined.

4.5. The effect of the choice of tag on overall success

Both York and Oxford routinely use vectors that introduce

short His6 purification tags. York has concentrated almost

exclusively on the non-cleavable N-terminal MGSSH-

HHHHH- tag, with a 3C protease-cleavable tag version (pET-

YSBLIC3C) currently undergoing assessment. Oxford

continues to explore cleavable and non-cleavable N-terminal

tags as well as cleavable C-terminal tags since this has been

found to increase the success rates for ‘difficult’ (i.e. non-

bacterial) targets as part of a multi-construct approach

(Siebold et al., 2005). Experience at both laboratories is that

longer tags adversely affect the ability to crystallize. From six

Oxford targets that produced satisfactory levels of soluble

expression with a long tag, only one gave crystals which were

of poor quality while the tag was still attached. After cleavage,

good-quality crystals could be grown for three of these targets.

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Figure 2Scatter plots of grand average of hydropathy (GRAVY; Kyte & Doolittle, 1982) against pI for proteins from B. anthracis. The background shading ofeach plot shows the clusters (A, B and C) identified from an analysis of T. maritima (Canaves et al., 2004). (a) A plot of proteins for all predictedB. anthracis ORFs. (b) A plot of the proteins selected by the York (triangles) and Oxford (circles) laboratories for HTP studies. The largely complete setsof 48 targets are shown as solid shapes, while those for which work is still in progress are shown as outline shapes. (c) A plot of targets for whichsignificant progress has been made at York (triangles) and Oxford (circles). Red (X-ray) and blue (NMR) shapes show structures that have beendetermined; yellow shapes show proteins which have not had structures determined but have yielded crystals; green shapes show proteins which have notgiven crystals but been expressed solubly in sufficient quantities to enter crystallization trials.

In general, it appears that the short N-terminal non-cleavable

tags used extensively by both York and Oxford have not

markedly impaired success rates for crystallization; however, a

quantitative assessment of the effect on crystal quality of the

short tag introduced by the pET-YSBLIC vector is ongoing in

York. Oxford has already generated a cohort of results (N = 13)

to help address the question of whether removing the His tag

is necessary. In four cases new or improved crystals were

obtained after tag cleavage, whilst in only two cases were

crystals better with an uncleaved tag. On the basis of these

results, the Oxford strategy is now to work with cleavable tags

that are as short as possible (both N-terminal 3C cleavable

tags and C-terminal carboxypeptidase cleavable tags). It is our

experience that the presence of the cleavage site does not of

itself significantly reduce protein expression or solubility

compared with a minimal non-cleavable tag.

4.6. Are target-selection strategies justified?

Oxford achieved a success rate of 31% for 48 targets by

repeating efforts and refining protocols, whereas York

achieved a success rate of 21% from the completed set of 48

targets but made fewer attempts at target rescue. Groups of

proteins within the B. anthracis targets showed particular

characteristics. For example, of the 111 York targets entered

into crystallization trials, 19 (17%) represented TIGR-

annotated (http://www.tigr.org) functional categories involved

in purine, pyrimidine, nucleoside and nucleotide metabolism,

the largest single category of proteins in crystallization. All 19

of these targets produced crystals (of varying quality) with

nine leading to structure solution, making this family parti-

cularly crystal-friendly. In contrast, the tRNA synthetases

tackled by Oxford proved extremely difficult to crystallize

despite giving adequate yields of soluble protein and only one

structure (at 2.8 A resolution) was obtained. This experience

reflects the general difficulty of working with tRNA synthe-

tases. As exemplified by the tRNA synthetases, where several

members of a single protein family were tackled in Oxford, at

least one structure was obtained for every family for which

multiple members were targeted (six families)2 supporting the

strategy of selecting paralogues in order to increase overall

success. However, it is interesting to note that the several

conserved hypothetical proteins that were included because

they are widely conserved across bacteria proved difficult. In

fact, only one out of four has yielded a structure to date.

Finally, the B. anthracis targets demonstrate that the use of

disorder-prediction methods, such as RONN and PONDR

(see Esnouf et al., 2006) have utility; an example of this is

provided by work on a conserved hypothetical protein

(BA0541) where redesign of the construct, omitting 18 resi-

dues at the N-terminus, yielded a 3.3 A structure. This struc-

ture was the only hypothetical structure solved, but it is

notable that unlike the other hypothetical proteins, it

belonged to an established fold.

4.7. Comparison with a structural genomics study ofThermotoga maritima

The combined York/Oxford study of proteins from

B. anthracis has been on a much smaller scale than the full-

genome study of T. maritima performed by the JCSG (http://

www.jcsg.org). For that study, 1878 ORFs were cloned and

have so far resulted in 131 structure determinations. An

analysis of the correlates of success with physico-chemical

properties has been made for the T. maritima project

(Canaves et al., 2004) and it appears that some of the findings

apply to our B. anthracis cohort. In particular, when analysed

in terms of the calculated pI and grand average of hydropathy

index, the open reading frames of B. anthracis group in a

similar way to those of T. maritima (Fig. 2a). However, the C

cluster is sparsely populated and the B cluster does not extend

to such high pI values. Although these clusters were not

considered formally in target selection, it can be seen that both

York and Oxford tended strongly to select targets within

clusters A and B (Fig. 2b). In line with the findings of the

JCSG, the targets that led to successful structure determina-

tions were far more likely to come from cluster A (Fig. 2c). It

is intriguing to note that York did manage to solve two

structures from cluster B by NMR analysis. Such plots may

therefore be useful as a direct aid to target selection and

construct design. Overall, the percentage of targets selected by

York or Oxford that yielded structures was higher than that

obtained by the JCSG for their comprehensive attack on

T. maritima, suggesting that the Oxford and York target

selection strategies used were of value in eliminating intract-

able targets.

5. Conclusions

The York and Oxford studies on B. anthracis have yielded

much more than just structures, although the �30% success

rate they have achieved alone makes them very successful

studies. They have provided a test bed for many of the tech-

nological developments in these two laboratories, develop-

ments which have been shared across Europe, with

dissemination largely enabled by the SPINE project. The work

has driven the development of much improved vectors for

ligation-independent cloning strategies, has helped to

optimize expression and purification protocols, has further

validated the HTP nanolitre-scale crystallization methods, has

demonstrated the utility of the target-selection tools devel-

oped at the start of SPINE and has illustrated ways in which

these can be further improved, e.g. by the more systematic use

of disorder-prediction methods and by considering the clusters

in hydropathy and pI. The requirement for conservation

across a series of genomes imposed by the OPPF selection

criteria also appears to have been beneficial in eliminating

difficult proteins, but excludes proteins characteristic of

B. anthracis that may play an important role in pathogenesis.

Structural genomics studies have provided an enormous

impetus for methods development in structural biology and

few laboratories are now untouched by their effects. The

emphasis in SPINE has been to apply these methods to

systems of biological interest, the ultimate aim being to solve

significant problems more effectively by the use of HTP and

parallel technologies. However, the experience with

B. anthracis makes it clear that novel technologies should be

benchmarked on more tractable targets to reveal techno-

logical inadequacies that might otherwise remain hidden

within the high attrition rate observed with more challenging

problems.

We would like to thank Professor Colin Harwood

(Newcastle) for providing genomic DNA. We also thank the

staff of BM14 (Grenoble), the SRS (Daresbury), the ESRF

and EMBL (Grenoble) for access to synchrotrons and help

with data collection. This work was supported by the

European Commission as part of SPINE (Structural Proteo-

mics In Europe) contract No. QLG2-CT-2002-00988 under the

Integrated Programme ‘Quality of Life and Management of

Living Resources’. The OPPF is also funded by the Medical

Research Council UK.

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